This invention pertains to fluorescent agents, instruments, and techniques for measurement of organ function, and, more specifically, for real-time measurement of organ function.
Acute renal failure (ARF), as a complication of multiple surgical, medical and obstetrical conditions, represents an important individual and public health problem. Early identification of patients at risk, with prompt elimination of potential insults, is a golden rule that has saved many lives. Unfortunately, despite close implementation of this rule, the disease still accounts for a large morbidity and mortality, with a survival rate of about 50%, a figure which has not substantially improved since 1950 (Butkus, D., Arch. Intern. Med., 143: 209-212, 1983). This poor outcome contrasts with the almost unique ability of the kidney to undergo virtually complete recovery of function following an episode of transient ischemia or toxin-induced cellular destruction. This discrepancy between mortality and the potential for reversibility emphasizes the need for a reconsideration of current diagnostic and therapeutic options with the goal of assuring complete recovery of organ function after an episode of ARF.
Because the clinical condition of patients with ARF is determined largely by prior health status and the nature of the specific insult that led to renal failure, any therapeutic approach used to treat ARF should be simultaneously oriented toward correcting the precipitating cause and the impaired organ function. Hypoperfusion of the kidney is the most frequently recognized single insult leading to ARF in the setting of trauma, surgery, hemorrhage, or dehydration (Kellen, M., S. Aronson, et al., Anesth. Analg., 78: 134-142, 1994; Hou, S., D. Bushinsky, et al., Am. J. Med., 74: 243-248, 1983). Continuous and precise monitoring of cardiopulmonary function in such acute settings has been available for many years and has undoubtedly helped to restore normal circulatory status in the critically ill patient. At the present time, however, monitoring of renal function is done with crude measurements such as urine output and plasma creatinine. Unfortunately, because of their lengthy resolution time (the time required to obtain a single measurement of renal function), none of these parameters can be used for real-time monitoring of renal function. For example, creatinine clearance measurements have a resolution of about 12 hours. By the time a patient""s ARF was recognized by this technique, it would be too late to treat the patient and have any hope of saving the kidney. The inadequacy of standard techniques for monitoring renal function during critical care is the most salient limitation for prevention of ARF and for the determination of an appropriate therapy to correct organ failure.
Measurements of glomerular filtration rate (GFR) can be made directly by micropuncture or indirectly by clearance methods. Although direct techniques have produced major contributions in our understanding of the production and regulation of the glomerular ultrafiltrate in laboratory animals, the invasive nature of the procedures renders them of questionable value in humans. Clearance techniques, on the other hand, are normally used to measure renal function in humans. However, because the techniques have such lengthy resolution times, it is quite difficult to detect rapid changes in GFR that may occur under different physiological and pathological conditions. For instance, GFR changes during exercise (Barclay, J., W. Cooke, et al., J. Physiol. (London), 104: 14, 1946), with orthostatic hypotension (Papper, E. and S. Ngai, Ann. Rev. Med., 7: 213-224, 1956), and with changes in posture (Werko, L., H. Bucht, et al., Scand. J. Clin. Lab. Invest., 1: 321, 1949). The changes in GFR during exercise were only detected when the exercise level was very intense and the changes in cardiopulmonary function were quite persistent (Selkur, E., Handbook of Physiology: Circulation, J. Field, Ed. Washington, DC: Am. Physiol. Soc.,. Vol. 2, pp. 1457-1516, 1963). These results suggested that changes in GFR at low levels of exercise may have gone undetected due to the poor resolution time of the clearance techniques. In order to fully understand this important limitation of clearance techniques, one should ask how fast the changes in GFR might occur under an ideal experimental condition emulating a hypoperfusion event of the kidney. Studies performed in the isolated, perfused dog kidney indicate that sudden changes (within seconds) in perfusion pressure are very closely followed (also within a few seconds) by changes in GFR (Harvey, R., Circulation Res., 15: 178-182, 1964). Clearance techniques, on the other hand, have a totally different resolution time. It was recognized very early that a considerable interval (more than 30 minutes) is required for a sudden change in GFR to be initially detected in the composition of urine (Smith, H., The Kidney: Structure and Function in Health and Disease, New York: Oxford University Press, 1951). This time most likely represents the time required for the ultrafiltrate to pass down the tubules, collecting ducts, and ureters before it reaches and equilibrates with the urine already contained in the urinary bladder. Since at least two samples are needed to determine that the measurement is done at equilibrium, the minimal ideal resolution time for this procedure will be about 1 hour. This, plus the usual delay in measuring the concentration of an agent in urine and blood samples, represents a significant limitation in the use of this procedure for bedside, real-time, monitoring of renal function in patients with ARF.
Renal function has traditionally been measured by creatinine clearance. It is now recognized, however, that in addition to the technical problems with creatinine measurement and with urine collection, creatinine clearance is not an accurate measure of GFR (Carrie, B., H. Golbertz, et al., Am. J. Med., 69: 177-182, 1980; Price, M., J. Urol., 107: 339-340, 1972). Quantitative methods for measuring renal glomerular and tubular function with clearance techniques have been available for many years. The nonendogenously produced substance inulin probably meets the requirements of an ideal GFR agent (Smith, 1951). Although it has remained the xe2x80x9cgold standardxe2x80x9d , the chemical methods of measurement are unfortunately too cumbersome for routine use. In addition to seeking a substance that fulfills the requirements of a GFR agent, researchers have also sought to overcome the other major source of error in clearance measurements, namely, incomplete urine collection. Two approaches have been found to be successful. The most accurate, but technically difficult, is the constant infusion of a substance until an equilibrium is reached, at which point the plasma level is steady. The rate of infusion is then equal to the rate of loss in the urine and no urine collection is necessary (Earle, D. and R. Berliner, Proc. Soc. Exp. Biol. Med., 62: 262-264, 1946). Alternatively, the rate of plasma disappearance of a substance after a single intravenous injection is determined, enabling calculation of GFR (Sapirstein, L., D. Vidt, et al., Am. J. Physiol., 181: 330-336, 1955; Chantler, C. and T. Barratt, Archs. Dis. Child., 47: 613-617, 1972). The disappearance of the tracer is determined by taking multiple blood samples over a period of 3 to 4 hours and then measuring the radioactivity of the samples. In addition to the requirements that a GFR agent must be freely filtered by the glomerulus, four other basic criteria must apply if a substance is to be used to measure clearance without urine collection:
a. it must not be metabolized;
b. it must be cleared exclusively by glomerular filtration (no other route of excretion other than renal);
c. it must not be bound to plasma protein or extracellular components; and
d. it must not be reabsorbed by the nephron. 51Cr-EDTA, 99mTc-DTPA, and 125I-sodium iothalamate meet these requirements and are the accepted choices for measuring GFR in most clinical studies (Chantler, 1972; Sigman, E., C. Ellwood, et al., J. Nucl. Med., 7: 60-68, 1965). Most of these clearance techniques, although more accurate than creatinine clearance, have not been widely used because of their technical complexities. Moreover, all of these methods are grossly inadequate for real-time and accurate monitoring of renal function. Clearly, a method for real-time, accurate, and continuous measurement of GFR in acute clinical settings will be a tremendous help in the management of patients who have ARF or are at risk of developing ARF.
Because glomerular filtration is the first step in urine production, the measurement of GFR represents the most convenient and reliable parameter for evaluation of renal function. Although there is general agreement that inulin clearance is the best measure of GFR, there are, as indicated above, several inherent difficulties in the use of this agent. As an alternative to inulin, a number of agents labeled with radioactive tracers have been introduced in the past few years for the measurement of GFR, including several chelates such as 51Cr-EDTA (Stacy, B. and G. Thorburn, Science, 152: 1076-1078, 1966), 111mIn-DTPA (Reba, R., F. Hosain, et al., Radiology, 90: 147-152, 1968), 169Yb-DTPA (Hosain, F., R. Reba, et al., Int. J. AppL Radiat., 20: 517-524, 1969; Perrone, R., T. Stainman, et al., Am. J. Kidney Dis., 16: 224-235, 1990) and 140La-DTPA (Bianchi, C. and M. Blaufox, J. Nucl. Biol. Med., 12: 117-122, 1968). The introduction of a kit for rapid and simple preparation of 99mTc-DTPA has made this the most readily available agent used to measure GFR, either by blood clearance (Klopper, J., W. Hauser, et al., J. Nucl. Med., 13: 107-110, 1972; Barbour, G., C. Crumb, et al., J. Nucl. Med., 17: 317-320, 1976; Hilson, A., R. Mistry, et al., Br. J. Radiol, 49: 794-799, 1976) or by external detection (Blaufox, M., E. Potchen, et al., J. Nucl. Med., 8: 77-85, 1967; Cohen, M., J. Patel, et al., Pediatrics, 48: 377-391, 1971; Thirimurthi, K., M. Casey, et al., Nucl. Med. All. Sci., 28: 245-250, 1984).
Real-time monitoring of renal function: Taking advantage of this opportunity, we developed a new approach for noninvasive and real-time monitoring of renal function (Rabito, C., R. Moore, et al., J. Nucl. Med., 34: 199-207, 1993). The method is based on a variation of the single-injection technique (Donath, A., Acta. Pediatr. Scan., 60: 512-527, 1971), in which continuous and instantaneous measurement of radioactivity is performed with an external detector rather than with the intermittent and deferred assay of venous blood and is described more fully in our commonly owned patents, U.S. Pat. Nos. 5,647,363 and 5,301,673, the contents of which are incorporated herein by reference. A radiation detector attached to a miniature data logger was used to monitor the clearance of 99mTc-diethylene triamine pentaacetic acid (99mTc-DTPA) from the extracellular space (Rabito, 1993). After a short equilibration period, the system behaved as a compartment system with first order kinetics. In this system, the log of activity varies lineraly with time, with the rate constant given by the slope of the resulting line. Two important assumptions are involved in the use of this approach for monitoring renal function. The first assumption is that the measurement of the rate constant can be performed fast enough to approach real-time conditions. For instance, the slope or rate constant can be calculated from several consecutive measurements of activity performed for a few seconds during a short interval of only a few minutes. This rate constant can be updated every minute or less after entering each new individual measurement. The second assumption is that the measurement of the rate constant for the clearance of an xe2x80x9cidealxe2x80x9d glomerular filtration agent from the extracellular space constitutes a precise and reproducible estimate of GFR. In the single injection technique, GFR is usually calculated as the volume of distribution of the GFR agent multiplied by the rate constant. However, because the volume distribution is assumed to be constant after normalization by body surface area, the rate constant per se represents an accurate estimate of GFR. These assumptions are supported by the excellent correlation between the rate constant for clearance of 99mTc-DTPA and simultaneous GFR measurements performed with a standard 125I-iothalamate clearance technique in 50 patients with different degrees of renal dysfunction (FIG. 1).
Because of their limited resolution time, none of the current techniques to measure renal function could be used to demonstrate the improvement in the resolution time of this novel approach. As an alternative, we studied the response-time of the technique by using the device in patients undergoing a medical procedure with high incidence of ARF. The rationale was that, in the case of ARF, the instrument should closely follow any rapid change in the renal function that may take place during the event. Validation, however, could be performed by comparison with a standard clearance technique before and after the intervention and once the renal function has become stable. The procedure was used, for instance, to monitor renal function in patients at risk for ARF in the intensive care unit or during angiography. The results demonstrated that the technique detected rapid changes in renal function with a resolution time of as little as 2.5 to 5 min. (Rabito, 1993; Rabito, C., F. Panico, et al., J. Am. Soc. Nephrol., 4: 1421-1428, 1994; Rabito, C., L. Fang, et al., Radiology, 186: 851-854, 1993).
Although highly innovative, this technique has some limitations. First, due to strict regulations and high costs, not every physician or medical institution has access to the use of radioactive tracer techniques. Second, due to the risk of radiation exposure, the technique cannot be used to measure renal function during surgery, or in infants and pregnant or postpartum women. To overcome these limitations, we propose in this invention the development of fluorescent GFR and renal blood flow (RBF) agent to be used in conjunction with a fluorescence-activated renal monitor, to thereby eliminate the limitations of the current radioactive GFR and RBF agents.
It is an object of this invention to provide fluorescent agents to monitor specific functions in specific organs.
It is also an object of this invention to provide an instrument for standard and time-resolved transcutaneous fluorescence measurements of these agents.
It is a further object of this invention to provide a method for real-time measurement and monitoring of organ function determined from transcutaneous fluorescence measurements of these agents.
In one aspect, the invention is a method of detecting a clearance function in a subject. The method comprises providing an electroluminescent agent in a circulatory system of the subject, irradiating a tissue site with electromagnetic radiation having sufficient energy and intensity to be absorbed by the agent, detecting the intensity of emission from the tissue site, and repeating the step of detecting at known time intervals. The agent is not metabolized by the subject and is only cleared by a single mechanism. In addition, the agent does not bind plasma, protein, or extracellular components and is not reabsorbed by the subject. The method may further comprise irradiating the tissue site with a laser, for example, a pulsed laser. The step of repeating may be performed until elapsed time since the step of irradiating is about 90% of the decay time, for example, 50 ns or greater. After the step of detecting has been repeated a predetermined number of times, the step of irradiating may be repeated, and a background emission may have decayed to an insignificant level before the step of detecting is performed.
In one embodiment, the agent may be cleared exclusively by the glomerulus and may comprise a polyaminopolyacetic acid derivative conjugated with an electroluminescent moiety, which may comprise a lanthanide ion. The lanthanide ion may be trivalent and may comprise Ce+++, Nd+++, Sm+++, Eu+++, or Tb+++. The conjugate may exhibit fluorescence when irradiated with red or infrared light.
The polyaminopolyacetic acid derivative may be selected from diethylenetriaminepentaacetic acid (DTPA) ethylene glycol N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid (EGTA), or polyaminopolybis(2-aminoethyl ether) acetic acid. The polyaminopolyacetic acid derivative may comprise 
S may be a cyclic organic moiety having at least one oxygen or nitrogen atom, and R may be an organic functionality, for example, an acetate or a p-toluene sulfonyl group. S may be aromatic, aliphatic, substituted, or unsubstituted. For example, S may comprise a furanyl, tetrahydrofuranyl, pyrrolidinyl, furoyl, pyrrolyl, or substituted derivatives of these. Exemplary substituents include NO2, NH2, isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetomido, and carboxyl groups.
In another aspect, the invention is an apparatus for detection of a clearance rate of a substance from extracellular fluid. The apparatus comprises a light source capable of producing light having sufficient intensity and energy to be absorbed by an electroluminescent moiety in the subject""s extracellular fluid, an optical fiber to deliver light from the light source to the subject, a detector, an optical fiber to deliver light emitted by the electroluminescent moiety to the detector, and processing means to calculate the rate of depletion of the electroluminescent moiety based on values measured by the detector. The light source may be a pulsed laser having a frequency such that it emits light at a time interval which is a predetermined fraction of a decay time of the electroluminescent moiety.
In another aspect, the invention is an electroluminescent molecule. The molecule comprises a polyaminopolyacetic acid derivative conjugated with an electroluminescent moiety and exhibits fluorescence when irradiated with red or infrared light. The molecule may be attached to an antibody, a DNA fragment, an RNA fragment, an enzyme, or an enzyme co-factor attached to the polyaminopolyacetic acid derivative. The molecule may also include an oligonucleotide. In another embodiment, the invention is a method of performing magnetic resonance imaging on a patient. The method comprises injecting the electroluminescent molecule into a patient, exposing the patient to a magnetic field, exposing the patient to a radio frequency pulse, and measuring the emission of hydrogen ions within the patient after removal of the pulse.
In another aspect, the invention is a method of performing immunochemical analysis. The method comprises associating a first electroluminescent complex with an analyte, exposing the first electroluminescent complex to light at an absorbance wavelength of the complex, and detecting light emitted by the first electroluminescent complex. The first complex comprises a bicyclic polyaminopolyacetic acid analog and an electroluminescent agent chelated to the bicyclic polyaminopolyacetic acid analog. The electroluminescent agent may comprise a lanthanide ion. The lanthanide ion may be trivalent and may comprise Ce+++, Nd+++, Sm+++, Eu+++, or Tb+++. The method may further comprise associating a second ligand labeled with a second electroluminescent complex with a second analyte, wherein the emission wavelength of the second complex is detectably different from the emission wavelength of the first complex. The method may be performed with more than two ligands and complexes. The electroluminescent complex may exhibit a decay time greater than 50 ns. The steps of exposing and detecting may be repeated. The method may further comprise attaching a first ligand to the analyte, wherein the first electroluminescent complex is associated with the analyte via attachment to the ligand; alternatively, the first electroluminescent complex may be attached to the first ligand via a second ligand. The analyte may be immobilized on a support, for example, via a ligand. Association may comprise removing an electroluminescent agent associated with the analyte and coordinating the electroluminescent agent with the bicyclic polyaminopolyacetic acid analog to form the first electroluminescent complex. In this embodiment, the bicyclic polyaminopolyacetic acid analog is not attached to the analyte, and the electroluminescent agent is attached to the analyte via a ligand. The bicyclic polyaminopolyacetic acid analog may be sequestered in a micelle. The ligand may comprise an antibody, a DNA fragment, an RNA fragment, an enzyme, or an enzyme co-factor. The polyaminopolyacetic acid derivative may comprise 
S may be a cyclic organic moiety having at least one oxygen or nitrogen atom, and R may be an organic functionality, for example, an acetate or a p-toluene sulfonyl group. S may be aromatic, aliphatic, substituted, or unsubstituted. For example, S may comprise a furanyl, tetrahydrofuranyl, pyrrolidinyl, furoyl, pyrrolyl, or substituted derivatives of these. Exemplary substituents include NO2, NH2, isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetomido, and carboxyl groups.
In another aspect, the invention is a molecule comprising 
S may be a cyclic organic moiety having at least one oxygen or nitrogen atom, and R may be an organic functionality, for example, an acetate or a p-toluene sulfonyl group. S may be aromatic, aliphatic, substituted, or unsubstituted. For example, S may comprise a furanyl, tetrahydrofuranyl, pyrrolidinyl, furoyl, pyrrolyl, or substituted derivatives of these. Exemplary substituents include NO2, NH2, isothiocyanato, semicarbazido, thiosemicarbazido, maleimido, bromoacetomido, and carboxyl groups.